Local Production of Estrogen in Breast Cancer
The observation the 17b-estradiol (E2) levels are 10-fold higher in breast carcinoma tissue compared to plasma supports the role for intratumoral estrogen synthesis.1-2Recent studies have established that concentration gradients of aromatase expression occur within the breast, with the highest levels of expression occurring in sites proximal to a tumor. Aromatase, is an enzyme responsible for a key step in the biosynthesis of estrogens. It is a member of the cytochrome P450 superfamily (EC 188.8.131.52), which are monooxygenases that catalyze many reactions involved in steroidogenesis. In particular, aromatase is responsible for the aromatization of androgens into estrogens.
In the breast, benign or malignant epithelial cells lie in close contact with endothelial cell-lined capillaries, mesenchymal stromal cells (undifferentiated adipose fibroblasts also known as preadipocytes), and lipid-filled mature adipocytes. In breast adipose tissue, most aromatase (80–90%) expression is found in adipose fibroblasts rather than in mature adipocytes Normal breast adipose tissue maintains low levels of aromatase expression primarily via distal promoter I.4 and uses the proximally located promoters I.3 and II only minimally.It is postulated that once neoplastic cells start to replicate, tumor growth will be promoted by locally increased estrogen levels. In turn, growth factors produced by the tumor in response to locally increased estrogen levels may further increase aromatase expression in the surrounding adipose tissue.3
Thus, a positive feed-back loop is established in which locally-produced estrogens and tumor-derived growth factors act by paracrine and autocrine mechanisms to sustain the growth and development of the tumor. Additional support for this concept comes from the observation that aromatase expression in breast adipose tissue is regulated by enhancer elements that appear to respond positively to growth factors, in contrast to expression in granulosa cells, which are inhibited by growth factors.
Paracrine interactions between malignant breast epithelial cells, proximal adipose fibroblasts, and vascular endothelial cells are responsible for estrogen biosynthesis and the lack of adipogenic differentiation in breast cancer tissue. It seems that malignant epithelial cells secrete factors that inhibit the differentiation of surrounding adipose fibroblasts (preadipocytes) to mature adipocytes and stimulate aromatase expression in these undifferentiated adipose fibroblasts.4The in vivo presence of malignant epithelial cells also enhances aromatase expression in endothelial cells in breast tissue.5
The desmoplastic reaction (formation of the dense fibroblast layer surrounding malignant epithelial cells) is essential for structural and biochemical support for tumor growth. In fact, pathologists refer to 70% of breast carcinomas as “scirrhous”-type, indicating the rock-like consistency of these tumors.6This consistency comes from the tightly packed undifferentiated adipose fibroblasts around malignant epithelial cells.
In breast cancer malignant epithelial cells enrich the population of adipose fibroblasts by secreting large amounts of cytokines such as tumor necrosis factor (TNF) aand interleukin II (IL-II) to inhibit the differentiation of fibroblasts (preadipocytes) to mature adipocytes; thus, creating a dense fibroblast layer surrounding malignant epithelial cells (the desmoplastic reaction). As a result, the amount of Promotor I.4 specific aromatase transcript is increased in breast cancer tissue. More importantly, malignant breast epithelial cells secrete prostaglandin E2 (PGE2) and other unknown factors to cause aromatase promoter switching from I.4 to the more potent I.3 and II promoters of adipose fibroblasts, leading to an increase production of aromatase. In this way large numbers of these estrogen-producing cells are maintained proximal to malignant cells.4-5Epithelial-stromal interactions in a breast tumor enhance estrogen formation, inhibition of adipogenic differentiation, and angiogenesis. Estradiol (E) up-regulates formation of antiadipogenic cytokines (TNF and IL-11) in epithelial cells. These cytokines inhibit adipogenic transcription factors C/EBPα and PPARγ to block adipogenesis and thus enhance accumulation of undifferentiated fibroblasts that express aromatase. Epithelial cells secrete PGE2 and other factors that induce binding of the transcription factors LRH-1, phosphorylated ATF-2, and C/EBPβ to promoters I.3 and II to up-regulate aromatase expression in adipose fibroblasts. Since the aromatase promoter I.7 activity is also elevated in breast tumor tissue, it is speculated that the transcription factor GATA-2 up-regulates this promoter in increased numbers of endothelial cells as a result of enhanced angiogenesis in breast cancer. (Adapted from Bulun, S.E., Lin Z., Imir G. et al. Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Diseases: From Bench to Treatment. Pharmacologic Reviews. 2005;57(3):359-383.)
Malignant epithelial cells induce aromatase via activation of aberrant promoters in breast cancer tissue and adipose fibroblasts proximal to tumor. In breast adipose tissue, most aromatase (80-90%) expression is found in adipose fibroblasts rather than mature adipocytes. The breast adipose tissue in disease-free women maintains low levels of aromatase expression primarily via promoter I.4 that lies 73 kb upstream of the common coding region.20The proximal promoters I.3 and II are used only minimally in normal breast adipose tissue.
Transcription via activity of promoters II and I.3 in the breast tumor fibroblasts and malignant epithelial cells, however, is strikingly increased.8-11In addition, breast endothelial cells, which proliferate in the pro-angiogenic environment of of breast cancer, appear to be a significant site of aromatase expression via promoter I.7.12Thus, it seems that the prototype estrogen-dependent malignancy breast cancer takes advantage of four promoters used in various cell types for aromatase expression. The sum of aromatase mRNA species arising from these four promoters markedly increases total aromatase mRNA levels in breast cancer compared with the normal breast that uses almost exclusively promoter I.4. Thus, the paracrine interaction between malignant epithelial cells affects adipogenic differentiation and activates a subset of aromatase promoters to drive local estrogen production.
Alternative promoter use for aromatase expression in normal and malignant breast tissues. Normal breast adipose tissue maintains low levels of aromatase expression primarily via promoter I.4. Promoters I.3 and II are used only minimally in normal breast adipose tissue, whereas promoter I.3 and II activity in breast cancer are strikingly increased. Additionally, the endothelial-type promoter I.7 is upregulated in breast cancer. Thus, the levels of total aromatase mRNA levels from four promoters (II, I.3, I.7 and I.4) in breast cancer tissue are strikingly higher than normal breast tissue. (Adapted from Zhao, H., Zhou, L., Shangguan, A., J., Bulun, S., E. Aromatase expression and regulation in breast and endometrial cancer. J Mol Endocrinol. 2016 Jul; 57(1): R19-R33.)
Mechanisms giving rise to increased local concentrations of estrogen in breast cancer via aromatase overexpression within the tumor tissue have been demonstrated by a number of investigators. These studies showed strikingly increased local levels of estrone, estrone sulfate, and estradiol in breast tumor tissue compared with circulating estrogen levels.13-15
A series of paracrine interactions between malignant breast epithelial cells and surrounding adipose stroma were uncovered and explained increased local estrogen levels in the breast bearing a cancer. For example, independent studies from at least six different laboratories indicated striking increases in aromatase enzyme activity and mRNA levels in breast fat adjacent to cancer compared with those in distal fat or disease-free breast adipose tissue.3,8,10,11,16,17,18 Interestingly, the overall aromatase expression in breast adipose tissue in mastectomy specimens bearing a breast tumor was significantly higher than that in benign breast tissue removed for reduction mammoplasty.9,19,20
Tissue sources of estrogen in postmenopausal breast cancer. This figure exemplifies the important pathologic roles of extra-ovarian (peripheral) and local estrogen biosynthesis in an estrogen-dependent disease in postmenopausal women. The estrogen precursor androstenedione (A) originates primarily from the adrenal in the postmenopausal woman. Aromatase expression and enzyme activity in extra-ovarian tissues such as fat increases with advancing age. The aromatase activity in skin and subcutaneous adipose fibroblasts gives rise to formation of systemically available estrone (E1) and to a smaller extent estradiol (E2). The conversion of circulating A to E1 in undifferentiated breast adipose fibroblasts compacted around malignant epithelial cells and subsequent conversion of E1 to E2 in malignant epithelial cells provide high tissue concentrations of E2 for tumor growth. The clinical relevance of these findings is exemplified by the successful use of aromatase inhibitors to treat breast cancer. (Adapted from Bulun, S.E., Lin Z., Imir G. et al. Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Diseases: From Bench to Treatment. Pharmacologic Reviews. 2005;57(3):359-383.)
- Bonadonna, G., Hortobagyi G.H., Valagussa, P. Textbook of Breast Cancer, A clinical guide to therapy (Third Edition) Chapter 10: 206
- Thijssen, J. H., Blankenstein M.A. Endogenous oestrogens in normal and malignant endometrial and and mammary tissue. Eur J Cancer Clin Oncol. 1989;25(12):1953-1959.
- Bulun, S. E., Mahendroo M. S., Simpson E. R. Aromatase gene expression in adipose tissue: Relationship to Breast Cancer. The Journal of Steroid Biochemistry and Molecular Biology. 1994;49(4-6): 319-326.
- Meng L, Zhou J, Hironobu S, Suzuki T, Zeitoun K, and Bulun S (2001) TNFalpha and IL-11 secreted by malignant breast epithelial cells inhibit adipocyte differentiation by selectively downregulating C/EBPalpha and PPARgamma: mechanism of desmoplastic reaction. Cancer Res61: 2250-2255
- Shozu M, Sebastian S, Takayama K, Hsu WT, Schultz RA, Neely K, Bryant M, and Bulun SE (2003b) Estrogen excess associated with novel gain-of-function mutations affecting the aromatase gene. N Engl J Med348: 1855-65.
- Haagensen CD (1986) Diseases of the Breast. W.B. Saunders Company, Philadelphia.
- Mahendroo MS, Mendelson CR, and Simpson ER (1993) Tissue-specific and hormonally-controlled alternative promoters regulate aromatase cytochrome P450 gene expression in human adipose tissue. J Biol Chem268: 19463-19470
- Harada N, Utsumi T, and Takagi Y (1993) Tissue-specific expression of the human aromatase cytochrome P450 gene by alternative use of multiple exons I and promoters and switching of tissue-specific exons I in carcinogenesis. Proc Natl Acad Sci USA90: 11312-11316.
- Agarwal VR, Bulun SE, Leitch M, Rohrich R, and Simpson ER (1996) Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J Clin Endocrinol Metab81: 3843-3849.
- Utsumi T, Harada N, Maruta M, and Takagi Y (1996) Presence of alternatively spliced transcripts of aromatase gene in human breast cancer. J Clin Endocrinol Metab81: 2344-2349.
- Zhou C, Zhou D, Esteban J, Murai J, Siiteri PK, Wilczynski S, and Chen S (1996) Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcription-polymerase chain reaction. J Steroid Biochem Mol Biol59: 163-171.
- Sebastian S, Takayama K, Shozu M, and Bulun S (2002) Cloning and characterization of a novel endothelial promoter of the human CYP19 (aromatase P450) gene that is up-regulated in breast cancer tissue. Mol Endocrinol16: 2243-2254.
- Van Landeghem AAJ, Poortman J, Nabuurs M, and Thijssen JHH (1985) Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue.Cancer Res 45: 2907-2912.
- Chetrite G, Cortes-Prieto J, Philippe J, Wright F, and Pasqualini J (2000) Comparison of estrogen concentrations, estrone sulfatase and aromatase activities in normal and in cancerous, human breast tissues.J Steroid Biochem Mol Biol 72: 23-7.
- Geisler J, Berntsen H, and Lonning P (2000) A novel HPLC-RIA method for the simultaneous detection of estrone, estradiol and estrone sulphate levels in breast cancer tissue.J Steroid Biochem Mol Biol 72: 259-264
- O’Neill JS, Elton RA, and Miller WR (1988) Aromatase activity in adipose tissue from breast quadrants: a link with tumor site.Br Med J 296: 741-743.
- Reed MJ, Topping L, Coldham NG, Purohit A, Ghilchik MW, and James VH (1993) Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors.J Steroid Biochem Mol Biol 44:589-596.
- Sasano H, Nagura H, Harada N, Goukon Y, and Kimura M (1994) Immunolocalization of aromatase and other steroidogenic enzymes in human breast disorders.Hum Pathol 25: 530-535.
- Agarwal VR, Bulun SE, and Simpson ER (1995) Quantitative detection of alternatively spliced transcripts of the aromatase cytochrome P450 (CYP19) gene in aromatase-expressing human cells by competitive RT-PCR.Mol Cell Probes 9: 453-464.
Agarwal VR, Bulun SE, and Simpson ER (1995) Quantitative detection of alternatively spliced transcripts of the aromatase cytochrome P450 (CYP19) gene in aromatase-expressing human cells by competitive RT-PCR. Mol Cell Probes 9: 453-464.